Her2 IHC on coverslips - (Apr/16/2005 )

Any good protocol to do Her2 IHC on non paraffin embedded blocks.I am doing it on coverslips.Thanks-PAN

-nanavatip-

I would fix your cells in 1% PFA in PBS for 15 minutes and proceed with a standard IHC protocol using one of the well-established anti-human HER2 antibodies available from DAKO or a similar company. Message me if you need more info (not sure how detailed a protocol you need).

-MaximinaNYC-

The Her2 prim. antibody binds from the inside of the cell membrane so there should be permeabilization step. i am thinking of using TritonX100. Do you think this will help.

QUOTE (nanavatip @ Apr 16 2005, 11:10 PM)

Any good protocol to do Her2 IHC on non paraffin embedded blocks.I am doing it on coverslips.Thanks-PAN

-nanavatip-

Well if your anitbody is against the intracellular portion you NEED to permeabilize with some sort of detergent. What kind depends on the cells. IT should be gentle as possible yet thorough. Sometimes Triton-X 100 is too much and cells come off the slides - try it out at varying concentrations to see what works. But I ask why are you using an Ab that detects the intracellular portion? There are several Abs which bind the extracellular domain quite well and then no perm is needed -- makes life easy

QUOTE (nanavatip @ Apr 17 2005, 09:12 AM)

The Her2 prim. antibody binds from the inside of the cell membrane so there should be permeabilization step. i am thinking of using TritonX100. Do you think this will help.